The present invention relates to a recombinant antibody molecule (RAM), and especially a humanised antibody molecule (HAM), having specificity for an antigen present in the T-cell receptor-CD3 complex of most T-cells, to a process for its production using recombinant DNA technology and to its therapeutic use.
In the present application, the term xe2x80x9crecombinant antibody moleculexe2x80x9d (RAM) is used to describe an antibody produced by an process involving the use of recombinant DNA technology, including any analogues of natural immunoglobulins or their fragments. The term xe2x80x9chumanised antibody moleculexe2x80x9d (HAM) is used to describe a molecule having an antigen binding site derived from an immunoglobulin from a non-human species, remaining immunoglobulin-derived parts of the molecule being derived from a human immunoglobulin. The antigen binding site may comprise either complete variable domains fused onto constant domains or one or more complementarity determining regions grafted onto appropriate framework regions in the variable domains. The abbreviation xe2x80x9cMAbxe2x80x9d is used to indicate a monoclonal antibody.
In the description, reference is made to a number of publications by number. The publications are listed in numerical order at the end of the description.
Natural immunoglobulins have been known for many years, as have the various fragments thereof, such as the Fab, (Fabxe2x80x2)2 and Fc fragments, which can be derived by enzymatic cleavage. Natural immunoglobulins comprise a generally Y-shaped molecule having an antigen-binding site towards the end of each upper arm. The remainder of the structure, and particularly the stem of the Y, mediates the effector functions associated with immunoglobulins.
Natural immunoglobulins have been used in assay, diagnosis and, to a more limited extent, therapy. However, such uses, especially in therapy, have been hindered by the polyclonal nature of natural immunoglobulins. A significant step towards the realisation of the potential of immunoglobulins as therapeutic agents was the discovery of techniques for the preparation of monoclonal antibodies of defined specificity (ref. 1). However, most MAbs are produced by fusions of rodent spleen cells with rodent myeloma cells. They are therefore essentially rodent proteins. There are very few reports of the production of human MAbs.
Since most available MAbs are of rodent origin, they are naturally antigenic in humans and thus can give rise to an undesirable immune response termed the HAMA (Human Anti-Mouse Antibody) response. Therefore, the use of rodent MAbs as therapeutic agents in humans is inherently limited by the fact that the human subject will mount an immunological response to the MAb and will either remove it entirely or at least reduce its effectiveness. Thus, in practice, MAbs of rodent origin may not be used in patients for more than one or a few treatments as a HAMA response soon develops rendering the MAb ineffective as well as giving rise to undesirable reactions.
Proposals have therefore been made for making non-human MAbs less antigenic in humans. Such techniques can be generically termed xe2x80x9chumanisationxe2x80x9d techniques. These techniques generally involve the use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the antibody molecule.
Early methods for humanising MAbs involved production of chimeric antibodies in which an antigen binding site comprising the complete variable domains of one antibody is linked to constant domains derived from another antibody. Methods for carrying out such chimerisation procedures are described in EPO120694 (Celltech Limited), EP0125023 (Genetech Inc. and City of Hope), EP-A-0 171496 (Res. Dev. Corp. Japan), EP-A-0 173 494 (Stanford University), and WO 86/01533 (Celltech Limited). This latter Celltech application (WO 86/01533) discloses a process for preparing an antibody molecule having the variable domains from a mouse MAb and the constant domains from a human immunoglobulin. Such humanised chimeric antibodies, however, still contain a significant proportion of non-human amino acid sequence, i.e. the complete non-human variable domains, and thus may still elicit non-human variable domains, and thus may still elicit some HAMA response, particularly if administered over a prolonged period [Begent al al Br. J.Cancer, 62: 487 (1990)].
WO 86/01533 also describes the production of an antibody molecule comprising the variable domains of a mouse MAb, the CHl and CL domains of a human immunoglobulin, and a non-immunoglobulin-derived protein in place of the Fc portion of the human immunoglobulin.
In an alternative approach, described in EP-A-0239400 (Winter), the complementarity determining regions (CDRs) of a mouse MAb have been grafted onto the framework regions of the variable domains of a human immunoglobulin by site directed mutagenesis using long oligonucleotides. There are 3 CDRs (CDR1, CDR2 and CDR3) in each of the heavy and light chain variable regions. Such CDR-grafted humanised antibodies are much less likely to give rise to a HAMA response than humanised chimeric antibodies in view of the much lower proportion of non-human amino acid sequence which they contain.
The earliest work on humanising MAbs by CDR-grafting was carried out on MAbs recognising synthetic antigens, such as the NP or NIP antigens. However, examples in which a mouse MAb recognising lysozyme and a rat MAb recognising an antigen on human T-cells respectively were humanised by CDR-grafting are shown by Verhoeyen et al (ref. 2) and Riechmann et al (ref. 3). The preparation of CDR-grafted antibody to the antigen on human T cells is also described in WO 89/07452 (Medical Research Council).
In Riechmann et al it was found that transfer of the CDR regions alone (as defined by Kabat refs. 4 and 5) was not sufficient to provide satisfactory antigen binding activity in the CDR-grafted product. Riechmann et al found that it was necessary to convert a serine residue at position 27 of the human sequence to the corresponding rat phenylalanine residue to obtain a CDR-grafted product having satisfactory antigen binding activity. This residue at position 27 of the heavy chain is within the structural loop adjacent to CDR1. A further construct which additionally contained a human serine to rat tyrosine change at position 30 of the heavy chain did not have a significantly altered binding activity over the humanised antibody with the serine to phenylalanine change at position 27 alone. These results indicate that changes to residues of the human sequence outside the CDR regions, in particular in the loop adjacent to CFR1, may be necessary to obtain effective antigen binding activity for CDR-grafted antibodies which recognise more complex antigens. Even so the binding affinity of the best CDR-grafted antibodies obtained was still significantly less than the original MAb.
Very recently Queen et al (ref. 6) have described the preparation of a humanised antibody that binds to the interleukin 2 receptor, by combining the CDRs of a murine MAb (anti-Tac) with human immunoglobulin framework and constant regions. The human framework regions were chosen to maximise homology with the anti-Tac MAb sequence. In addition computer modelling was used to identify framework amino acid residues which were likely to interact with the CDRs or antigen, and mouse amino acids were used at these positions in the humanised antibody.
In WO 90/07861 Queen et al propose four criteria for designing humanised immunoglobulins. The first criterion is to use as the human acceptor the framework from a particular human immunoglobulin that is unusually homologous to the non-human donor immunoglobulin to be humanised, or to use a consensus framework from many human antibodies. The second criterion is to use the donor amino acid rather than the acceptor if the human acceptor residue is unusual and the donor residue is typical for human sequences at a specific residue of the framework. The third criterion is to use the donor framework amino acid residue rather than the acceptor at positions immediately adjacent to the CDRs. The fourth criterion is to use the donor amino acid residue at framework positions at which the amino acid is predicted to have a side chain atom within about 3 xc3x85 of the CDRs in a three-dimensional immunoglobulin model and to be capable of interacting with the antigen or with the CDRs of the humanised immunoglobulin. It is proposed that criteria two, three of four may be applied in addition or alternatively to criterion one, and may be applied singly or in any combination.
WO 90/07861 described in detail the preparation of a single CDR-grafted humanised antibody, a humanised antibody having specificity for the p55 Tac protein of the IL-2 receptor. The combination of all four criteria, as above, were employed in designing this humanised antibody, the variable region frameworks of the human antibody Eu (refs. 4 and 5) being used as acceptor. In the resultant humanised antibody the donor CDRs were as defined by Kabat et al (refs. 4 and 5) and in addition the mouse donor residues were used in place of the human acceptor residues, at positions 27, 30, 48, 66, 67, 89, 91, 94, 103, 104, 105 and 107 in the heavy chain and at positions 48, 60 and 63 in the light chain, of the variable region frameworks. The humanised anti-Tac antibody obtained is reported to have an affinity for p55 of 3xc3x97109 Mxe2x88x921, about one-third of that of the murine MAb.
OKT3 is a mouse IgG2a/k MAb which recognises an antigen in the T-cell receptor-CD3 complex and has been approved for use in many countries throughout the world as an immunosuppressant in the treatment of acute allograft rejection [Chatenoud et al (ref. 7), and Jeffers et al (ref. 8) However, in view of the murine nature of this MAb, a significant HAMA response, with a major anti-idiotype component, may build up on use. Clearly, it would be highly desirable to diminish or abolish this undesirable HAMA response by suitable humanisation or other recombinant DNA manipulation of this very useful antibody and thus enlarge its area of use. It would also be desirable to apply the techniques of recombinant DNA technology more generally to this useful antibody to prepare RAM products.
Moreover, we have further investigated the preparation of CDR-grafted humanised antibody molecules and have identified a hierarchy of positions within the framework of the variable regions (i.e. outside both the Kabat CDRs and structural loops of the variable regions) at which the amino acid identities of the residues are important for obtaining CDR-grafted products with satisfactory binding affinity. This has enabled us to establish a protocol for obtaining satisfactory CDR-grafted products which may be applied very widely irrespective of the level of homology between the donor immunoglobulin and acceptor framework. The set of residues which we have identified as being of critical importance does not coincide with the residues identified by Queen et al (ref. 6).
Accordingly the present invention provides an RAM comprising antigen binding regions derived from the heavy and/or light chain variable regions of a donor anti-CD3 antibody and having anti-CD3 binding specificity, and preferably having an anti-CD3 binding affinity similar to that of OKT3.
Typically the donor anti-CD3 antibody is a rodent MAb.
The RAM of the invention may comprise antigen binding regions from any suitable anti-CD3 antibody, typically a rodent anti-CD3 MAb, e.g. a mouse or rat anti-CD3 MAb. The RAM may comprise a recombinant version of whole or a major part of the amino acid sequence of such a MAb. Also the RAM may comprise only the variable region (VH and/or VL) or one or more CDRs of such a MAb. Especially the RAM may comprise amino acid sequences, whether variable region, CDR or other, derived from the specific anti-CD3 MAb (OKT3) hereinafter specifically described with reference to FIGS. 1 and 2.
Preferably the RAM of the present invention is a humanised antibody molecule (HAM) having specificity for CD3 and having an antigen binding site wherein at least one of the complementarity determining regions (CDRs) of the variable domain, usually at least two and preferably all of the CDRs, are derived from a non-human anti-CD3 antibody, e.g. a rodent anti-CD3 MAb.
The RAM may be a chimeric antibody or a CDR-grafted antibody.
Accordingly, in preferred embodiments the invention provides an anti-CD3 CDR-grafted antibody heavy chain having a variable region domain comprising acceptor framework and donor CD3 binding regions wherein the framework comprises donor residues at at least one of positions 6, 23 and/or 24, 48 and/or 49, 71 and/or 73, 75 and/or 76 and/or 78 and 88 and/or 91.
More preferably, the heavy chain framework of the preferred embodiment comprises donor residues at positions 23, 24, 49, 71, 73 and 78 or at positions 23, 24 and 49. The residues at positions 71, 73 and 78 of the heavy chain framework are preferably either all acceptor or all donor residues.
In particularly preferred embodiments the heavy chain framework additionally comprises donor residues at one, some or all of positions 6, 37, 48 and 94. Also it is particularly preferred that residues at positions of the heavy chain framework which are commonly conserved across species, i.e. positions 2, 4, 25, 36, 39, 47, 93, 103, 104, 106 and 107, if not conserved between donor and acceptor, additionally comprise donor residues. Most preferably the heavy chain framework additionally comprises donor residues at positions 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and 107.
In addition the heavy chain framework optionally comprises donor residues at one, some or all of positions:
1 and 3,
72 and 76,
69 (if 48 is different between donor and acceptor),
38 and 46 (if 48 is the donor residue),
80 and 20 (if 69 is the donor residue),
67,
82 and 18 (if 67 is the donor residue),
91,
88, and
any one or more of 9, 11, 41, 87, 108, 110 and 112.
In the preferred embodiments of the present invention described above and hereinafter, reference is made to CDR-grafted antibody products comprising acceptor framework and donor antigen binding regions. It will be appreciated that the invention is widely applicable to the CDR-grafting of anti-CD3 antibodies in general. Thus, the donor and acceptor antibodies may be anti-CD3 antibodies derived from animals of the same species and even same antibody class or sub-class. More usually, however, the donor and acceptor antibodies are derived from animals of different species. Typically, the donor anti-CD3 antibody is a non-human antibody, such as a rodent MAb, and the acceptor antibody is a human antibody.
In the CDR-grafted antibody products of the present invention, the donor CD3 binding region typically comprises at least one CDR from the donor antibody. Usually the donor antigen binding region comprises at least two and preferably all three CDRs of each of the heavy chain and/or light chain variable regions. The CDRs may comprise the Kabat CDRs, the structural loop CDRs or a composite of the Kabat and structural loop CDRs and any combination of any of these. Preferably, the antigen binding regions of the CDR-grafted heavy chain variable domain comprise CDRs corresponding to the Kabat CDRs at CDR2 (residues 50-65) and CDR3 (residues 95-100) and a composite of the Kabat and structural loop CDRs at CDR1 (residues 26-35).
The residue designations given above and elsewhere in the present application are numbered according to the Kabat numbering (refs. 4 and 5). Thus the residue designations do not always correspond directly with the linear numbering of the amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or CDR, of the basic variable domain structure. For example, the heavy chain variable region of the anti-Tac antibody described by Queen et al (ref. 6) contains a single amino acid insert (residue 52a) after reside 52 of CDR2 and a three amino acid insert (residues 82a, 82b and 82c) after framework residue 82, in the Kabat numbering. The correct Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a xe2x80x9cstandardxe2x80x9d Kabat numbered sequence.
The invention also provides in a further preferred embodiment a CDR-grafted antibody light chain having a variable region domain comprising acceptor framework and donor CD3 binding regions wherein the framework comprises donor residues at at least one of positions 1 and/or 3 and 46 and/or 47. Preferably the CDR grafted light chain of this preferred embodiment comprises donor residues at positions 46 and/or 47.
The invention also provides in a yet further preferred embodiment a CDR-grafted antibody light chain having a variable region domain comprising acceptor framework and donor CD3 binding regions wherein the framework comprises donor residues at at least one of positions 46, 48, 58 and 71.
More preferably in this latter embodiment, the framework comprises donor residues at all of positions 46, 48, 58 and 71.
In particularly preferred embodiments of the above preferred embodiments, the light chain framework additionally comprises donor residues at positions 36, 44, 47, 85 and 87. Similarly positions of the light chain framework which are commonly conserved across species, i.e. positions 2, 4, 6, 35, 49, 62, 64-69, 98, 99, 101 and 102, if not conserved between donor and acceptor, additionally comprise donor residues. Most preferably the light chain framework additionally comprises donor residues at positions 2, 4, 6, 35, 36, 38, 44, 47, 49, 62, 64-69, 85, 87, 98, 99, 101 and 102.
In additions the light chain framework of the above preferred embodiments optionally comprises donor residues at one, some or all of positions:
1 and 3,
63,
60 (if 60 and 54 are able to form at potential saltbridge),
70 (if 70 and 24 are able to form a potential saltbridge),
73 and 21 (if 47 is different between donor and acceptor),
37 and 45 (if 47 is different between donor and acceptor), and
any one or more of 10, 12, 40, 80, 103 and 105.
Preferably, the antigen binding regions of the CDR-grafted light chain variable domain comprise CDRs corresponding to the Kabat CDRs at CDR1 (residue 24-34), CDR2 (residues 50-56) and CDR3 (residues 89-97).
The invention further provides in a fourth aspect a CDR-grafted antibody molecule comprising at least one CDR-grafted heavy chain and at least one CDR-grafted light chain as defined above.
The CDR-grafted and humanised antibody molecules and chains of the present invention may comprise: a complete antibody molecule, having full length heavy and light chains; a fragment thereof, such as a Fab, (Fabxe2x80x2)2 or FV fragment; a light chain or heavy chain monomer or dimer; or a single chain antibody, e.g. a single chain FV in which heavy and light chain variable regions are joined by a peptide linker; or any other CDR-grafted or humanised antibody product with anti-CD3 binding specificity. Similarly the CDR-grafted heavy and light chain variable region may be combined with other antibody domains as appropriate.
Also the CDR-grafted or humanised heavy or light chains or antibody molecules of the present invention may have attached to them an effector or reporter molecule. For instance, it may have a macrocycle, for chelating a heavy metal atom, or a toxin, such as ricin, attached to it by a covalent bridging structure. Alternatively, the procedures of recombinant DNA technology may be used to produce an immunoglobulin molecule in which the Fc fragment of CH3 domain of a complete immunoglobulin molecule has been replaced by, or has attached thereto by peptide linkage, a functional non-immunoglobulin protein, such as an enzyme or toxin molecule.
For CDR-grafted antibody products, any appropriate acceptor variable region framework sequences may be used having regard to class/type of the donor antibody from which the antigen binding regions are derived. Preferably, the type of acceptor framework used is of the same/similar class/type as the donor antibody. Conveniently, the framework may be chosen to maximise/optimise homology with the donor antibody sequence particularly at positions close or adjacent to the CDRs. However, a high level of homology between donor and acceptor sequences is not important for application of the present invention. The present invention identifies a hierarchy of framework residue positions at which donor residues may be important or desirable for obtaining a CDR-grafted antibody product having satisfactory binding properties. The present invention advantageously enables the preparation of CDR-grafted antibody products having binding affinities similar to, and even in some cases better than the corresponding donor antibody product, e.g. OKT3 product. Preferably, the CDR-grafted antibody products of the invention have binding affinities of at least about 105 Mxe2x88x921, preferably at least about 108 Mxe2x88x921 and especially within the range 108xe2x88x921012 Mxe2x88x921. In principle, the present invention is applicable to any combination of donor and acceptor antibodies irrespective of the level of homology between their sequences. A protocol for applying the invention to any particular donor-acceptor antibody pair is given hereinafter. Examples of human frameworks which may be used are KOL, NEWM, REI, EU, LAY and POM (refs. 4 and 5); for instance KOL and NEWM for the heavy chain and RE1 for the light chain and EU, LAY and POM for both the heavy chain and the light chain.
Also the constant region domains of the products of the invention may be selected having regard to the proposed function of the antibody in particular the effector functions which may be required. For example, the constant region domains may be human IgA, IgE, IgG or IgM domains. In particular, IgG human constant region domains may be used, especially of the IgG1 and IgG3 isotypes, when the humanised antibody molecule is intended for therapeutic uses, and antibody effector functions are required. Alternatively, IgG2 and IgG4 isotypes may be used when the humanised antibody molecule is intended for therapeutic purposes and antibody effector functions are not required, e.g. for simple blocking of the T-cell receptor-CD3 complex.
However, the remainder of the antibody molecules need not comprise only protein sequences from immunoglobulins. For instance, a gene may be constructed in which a DNA sequence encoding part of a human immunoglobulin chain is fused to a DNA sequence encoding the amino acid sequence of a polypeptide effector or reporter molecule.
Preferably the CDR-grafted heavy and light chain and antibody molecule products are produced by recombinant DNA technology.
Thus in further aspects the invention also includes DNA sequences coding for the RAMs, HAMs and CDR-grafted heavy and light chains, cloning and expression vectors containing the DNA sequences, host cells transformed with the DNA sequences and processes for producing the CDR-grafted antibody molecules comprising expressing the DNA sequences in the transformed host cells.
The general methods by which the vectors may be constructed, transfection methods and culture methods are well known per se and form no part of the invention. Such methods are shown, for instance, in references 9 and 10.
The DNA sequences which encode the anti-CD3 donor amino acid sequences may be obtained by methods well known in the art. For example the anti-CD3 coding sequences may be obtained by genomic cloning, or cDNA cloning from suitable hybridoma cell lines, e.g. the OKT3 cell line hereinafter specifically described. Positive clones may be screened using appropriate probes for the heavy and light chain genes in question. Also PCR cloning may be used.
DNA coding for acceptor, e.g. human acceptor, sequences may be obtained in any appropriate way. For example DNA sequences coding for preferred human acceptor frameworks such as KOL, REI, EU and NEWM, are widely available to workers in the art.
The standard techniques of molecular biology may be used to prepare DNA sequences coding for the chimeric and CDR-grafted products. Desired DNA sequences may be synthesised completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate. For example oligonucleotide directed synthesis as described by Jones et al (ref. 17) may be used. Also oligonucleotide directed mutagenesis of a pre-exising variable region as, for example, described by Verhoeyen et al (ref. 2) or Riechmann et al (ref. 3) may be used. Also enzymatic filling in of gapped oligonucleotides using T4 DNA polymerase as, for example, described by Queen et al (ref. 6) may be used.
Any suitable host cell/vector system may be used for expression of the DNA sequences coding for the CDR-grafted heavy and light chains. Bacterial e.g. E. coli, and other microbial systems may be used, in particular for expression of antibody fragments such as FAb and (Fabxe2x80x2)2 fragments, and especially FV fragments and single chain antibody fragments e.g. single chain FVs. Eucaryotic, e.g. mammalian, host cell expression systems may be used, in particular, for production of larger CDR-grafted antibody products, including complete antibody molecules. Suitable mammalian host cells include CHO cells and myeloma or hybridoma cell lines.
Thus, according to a further aspect the present invention provides a process for producing an anti-CD3 RAM which process comprises:
(a) producing in an expression vector an operon having a DNA sequence which encodes an antibody heavy chain wherein at least one CDR of the variable domain is derived from a donor anti-CD3 antibody and remaining immunglobulin-derived parts of the antibody chain are derived from an acceptor immunoglobulin;
and/or
(b) producing in an expression vector an operon having a DNA sequence which encodes a complementary antibody light chain wherein at least one CDR of the variable domain is derived from a donor anti-CD3 antibody and the remaining immunoglobulin-derived parts of the antibody chain are derived from an acceptor immunoglobulin;
(c) transfecting a host cell with the or each vector;
and
(d) culturing the transfected cell line to produce the RAM.
The RAM may comprise only heavy or light chain-derived polypeptide, in which case only a heavy chain or light chain polypeptide coding sequence is used to transfect the host cells.
For production of RAMs comprising both heavy and light chains, the cell line may be transfected with two vectors. The first vector may contain an operon encoding a light chain-derived polypeptide and the second vector may contain an operon encoding a heavy chain-derived polypeptide. Preferably, the vectors are identical except in so far as the coding sequences and selectable markers are concerned so as to ensure as far as possible that each polypeptide chain is equally expressed. Alternatively, a single vector may be used, the vector including the sequences encoding both light chain- and heavy chain-derived polypeptides.
The DNA in the coding sequences for the light and heavy chains may comprise cDNA or genomic DNA or both. However, it is preferred that the DNA sequence encoding the heavy or light chain comprises, at least partially, genomic DNA. Most preferably, the heavy or light chain encoding sequence comprises a fusion of cDNA and genomic DNA.
The present invention also includes therapeutic and diagnostic compositions comprising the RAMs, HAMs and CDR-grafted light and heavy chains and molecules of the invention and uses of such compositions in therapy and diagnosis.
Accordingly in a further aspect the invention provides a therapeutic or diagnostic composition comprising a RAM, HAM or CDR-grafted antibody heavy or light chain or molecule according to previous aspects of the invention in combination with a pharmaceutically acceptable carrier, diluent or excipient.
Accordingly also the invention provides a method of therapy or diagnosis comprising administering an effective amount of a RAM, HAM or CDR-grafted antibody heavy or light chain or molecule according to previous aspects of the invention to a human or animal subject.
The RAM, HAM and CDR-grafted products of the present invention may be used for any of the therapeutic uses for which anti CD3 antibodies, e.g. OKT3, have been used or may be used in the future. For example, the products may be used as ummunosuppressants, e.g. in the treatment of acute allograft rejection.
A preferred protocol for obtaining CDR-grafted antibody heavy and light chains in accordance with the present invention is set out below together with the rationale by which we have derived this protocol. This protocol and rationale are given without prejudice to the generality of the invention as hereinbefore described and defined.
Protocol
It is first of all necessary to sequence the DNA coding for the heavy and light chain variable regions of the donor antibody, to determine their amino acid sequences. It is also necessary to choose appropriate acceptor heavy and light chain variable regions, of known amino acid sequence. The CDR-grafted chain is then designed starting from the basis of the acceptor sequence. It will be appreciated that in some cases the donor and acceptor amino acid residues may be identical at a particular position and thus no change of acceptor framework residue is required.
1. As a first step donor residues are substituted for acceptor residues in the CDRs. For this purpose the CDRs are preferably defined as follows:
The positions at which donor residues are to be substituted for acceptor in the framework are then chosen as follows, first of all with respect to the heavy chain and subsequently with respect to the light chain.
2. Heavy Chain
2.1 Choose donor residues at all of positions 23, 24, 49, 71, 73 and 78 of the heavy chain or all of positions 23, 24 and 49 (71, 73 and 78 are either all donor or all acceptor).
2.2 Check that the following have the same amino acid in donor and acceptor sequences, and if not preferably choose the donor: 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and 107.
2.3 To further optimise affinity consider choosing donor residues at one, some or any of:
i. 1, 3
ii. 72, 76
iii. If 48 is different between donor and acceptor sequences, consider 69
iv. If at 48 the donor residue is chosen, consider 38 and 46
v. If at 69 the donor residue is chosen, consider 80 and then 20
vi. 67
vii. If at 67 the donor residue is chosen, consider 82 and then 18
viii. 91
ix. 88
x. 9, 11, 41, 87, 108, 110, 112
3. Light Chain
3.1 Choose donor at 46, 48, 58 and 71
3.2 Check that the following have the same amino acid in donor and acceptor sequences, if not preferably choose donor: 2, 4, 6, 35, 38, 44, 47, 49, 62, 64-69 inclusive, 85, 87, 98, 99, 101 and 102
3.3 To further optimise affinity consider choosing donor residues at one, some or any of:
i. 1, 3
ii. 63
iii. 60, if 60 and 54 are able to form a potential saltbridge
iv. 70, if 70 and 24 are able to form a potential saltbridge
v. 73 and 21, if 47 is different between donor and acceptor
vi. 37 and 45, if 47 is different between donor and acceptor
vii. 10, 12, 40, 80, 103, 105
Rationale
In order to transfer the binding site of an antibody into a different acceptor framework, a number of factors need to be considered.
1. The extent of the CDRs
The CDRs (Complementary Determining Regions) were defined by Wu and Kabat (refs. 4 and 5) on the basis of an analysis of the variability of different regions of antibody variable regions. Three regions per domain were recognised. In the light chain the sequences are 24-34, 50-56, 89-97 (numbering according to Kabat (ref. 4), Eu Index) inclusive and in the heavy chain the sequences are 31-35, 50-65 and 95-102 inclusive.
When antibody structures became available it became apparent that these CDR regions corresponded in the main to loop regions which extended from the xcex2 barrel framework of the light and heavy variable domains. For H1 there was a discrepancy in that the loop was from 26 to 32 inclusive and for H2 to loop was 52-56 and for L2 from 50 to 53. However, with the exception of H1 the CDR regions encompassed the loop regions and extended into the xcex2 strand frameworks. In H1 residue 26 tends to be a serine and 27 a phenylalanine or tyrosine, residue 29 is a phenylalanine in most cases. Residues 28 and 30 which are surface residues exposed to solvent might be involved in antigen-binding. A prudent definition of the H1 CDR therefore would include residues 26-35 to include both the loop region and the hypervariable residues 33-35.
It is of interest to note the example of Riechmann et al (ref. 3), who used the residue 31-35 choice for CDR-H1. In order to produce efficient antigen binding residue 27 also needed to be recruited from the donor (rat) antibody.
2. Non-CDR residues which contribute to antigen binding
By examination of available X-ray structures we have identified a number of residues which may have an effect on net antigen binding and which can be demonstrated by experiment. These residues can be sub-divided into a number of groups.
2.1 Surface residues near CDR [all numbering as in Kabat et al (ref. 4)].
2.1.1. Heavy Chainxe2x80x94Key residues are 23, 71 and 73. Other residues which may contribute to a lesser extent are 1, 3 and 76. Finally 25 is usually conserved but the murine residue should be used if there is a difference.
2.1.2 Light Chainxe2x80x94Many residues close to the CDRs, e.g. 63, 65, 67 and 69 are conserved. If conserved none of the surface residues in the light chain are likely to have a major effect. However, if the murine residue at these positions is unusual, then it would be of benefit to analyse the likely contribution more closely. Other residues which may also contribute to binding are 1 and 3, and also 60 and 70 if the residues at these positions and at 54 and 24 respectively are potentially able to form a salt bridge i.e. 60+54; 70+24.
2.2 Packing residues near the CDRs.
2.2.1. Heavy Chainxe2x80x94Key residues are 24, 49 and 78. Other key residues would be 36 if not a tryptophan, 94 if not an arginine, 104 and 106 if not glycines and 107 if not a threonine. Residues which may make a further contribution to stable packing of the heavy chain and hence improved affinity are 2, 4, 6, 38, 46, 67 and 69. 67 packs against the CDR residue 63 and this pair could be either both mouse or both human. Finally, residues which contribute to packing in this region but from a longer range are 18, 20, 80, 82 and 86. 82 packs against 67 and in turn 18 packs against 82. 80 packs against 69 and in turn 20 packs against 80. 86 forms an H bond network with 38 and 46. Many of the mouse-human differences appear minor e.g. Leu-Ile, but could have an minor impact on correct pacing which could translate into altered positioning of the CDRs.
2.2.2 Light Chainxe2x80x94Key residues are 48, 58 and 71. Other key residues would be 6 if not glutamine, 35 if not tryptophan, 62 if not phenylalanine or tryosine, 64, 66, 68, 99 and 101 if not glycines and 102 if not a threonine. Residues which make a further contribution are 2, 4, 37, 45 and 47. Finally residues 73 and 21 and 19 may make long distance packing contributions of a minor nature.
2.3. Residues at the variable domain interface between heavy and light chainsxe2x80x94In both the light and heavy chains most of the non-CDR interface residues are conserved. If a conserved residue is replaced by a residue of different character, e.g. size or charge, it should be considered for retention as the murine residue.
2.3.1 Heavy Chainxe2x80x94Residues which need to be considered are 37 if the residue is not a valine but is of larger side chain volume or has a charge or polarity. Other residues are 39 if not a glutamine, 45 if not a leucine, 47 if not a tryptophan, 91 if not a phenylalanine or tyrosine, 93 if not an alanine and 103 if not a tryptophan. Residue 89 is also at the interface but is not in a position where the side chain could be of great impact.
2.3.2. Light Chainxe2x80x94Residues which need to be considered are 36, if not a tyrosine, 38 if not a glutamine, 44 if not a proline, 46, 49 if not a tyrosine, residue 85, residue 87 if not a tyrosine and 98 if not a phenylalanine.
2.4. Variable-Constant region interfacexe2x80x94The elbow angle between variable and constant regions may be affected by alterations in packing of key residues in the variable region against the constant region which may effect the position of VL and VH with respect to one another. Therefore it is worth noting the residues likely to be in contact with the constant region. In the heavy chain the surface residues potentially in contact with the variable region are conserved between mouse and human antibodies therefore the variable region contact residues may influence the V-C interaction. In the light chain the amino acids found at a number of the constant region contact points vary, and the V and C regions are not in such close proximity as the heavy chain. Therefore the influences of the light chain V-C interface may be minor.
2.4.1. Heavy Chainxe2x80x94Contact residues are 7, 11, 41, 87, 108, 110, 112.
2.4.2. Light Chainxe2x80x94In the light chain potentially contacting residues are 10, 12, 40, 80, 83, 103 and 105.
The above analysis coupled with our considerable practical experimental experience in the CDR-grafting of a number of different antibodies have lead us to the protocol given above.
The present invention is now described, by way of example only, with reference to the accompanying FIGS. 1-13.